Trophic Conversion of Obligate Phototrophic Algae Through Metabolic Engineering

ABSTRACT

Most microalgae are obligate photoautotrophs and their growth is strictly dependent on the generation of photosynthetically-derived energy. In this study it is shown that the microalga  Phaeodaclylurn tricornutum  can be engineered to import glucose and grow in the dark through the introduction of genes encoding glucose transporters. Both the human and  Chlorella kessleri  glucose transporters facilitated the uptake of glucose by  P. tricornutum , allowing the cells to metabolize exogenous organic carbon and thrive, independent of light. This is the first successful trophic conversion of an obligate photoautotroph through metabolic engineering, and it demonstrates that methods of cell nourishment can be fundamentally altered with the introduction of a single gene. Since strains transformed with the glucose transport genes are able to grow non-photosynthetically, they can be exploited for the analysis of photosynthetic processes through mutant generation and characterization. Finally, this work also represents critical progress toward large-scale commercial exploitation of obligate phototrophic algae through the use of microbial fermentation technology, eliminating significant limitations resulting from light-dependent growth.

This application is based on U.S. provisional application No.60/198,742, filed Apr. 21, 2000, which is incorporated herein in itsentirety.

This work was supported by National Science Foundation Small BusinessInnovation Research Grant #9710990. The U.S. Government may have certainrights in this invention.

BACKGROUND

1. Field of the Invention

This invention is directed to methods for genetic transformation ofalgae, and in particular to conversion of obligate phototrophicorganisms to recombinant organisms capable of heterotrophic growth.

2. Review of Related Art

Photosynthetic algae are the primary producers in aquatic environments,accounting for a significant proportion of worldwide O₂ production andCO— fixation in aquatic environments. Tréquer, P., Nelson, D. M., VanBennekom, A. J., DeMaster, D. J., Leynaert, A. & Quéquiner, B. 1995.“The silica balance in the world ocean: a reestimate,” Science269:375-79. Algae—are also needed for aquaculture and are used toproduce many valuable products. For example, algae are used for theproduction of pigments (e.g., β-carotene, phycobiliproteins), oils withnutritional value (e.g., docosahexaenoic acid), and stableisotope-labeled biochemicals (e.g., ¹³C-glucose). Algae are also used asfood for human and animal consumption.

In general, algae require light to drive photosynthesis for theproduction of the chemical energy required for cellular metabolism. Manyare obligate phototrophs, meaning they have an absolute requirement forlight to survive. Droop M R “Heterotrophy of Carbon.” in AlgalPhysiology and Biochemistry Botanical Monographs, 10: 530-559, ed.Stewart W D P, University of California Press, Berkeley (1974). Suchalgae are unable to utilize exogenous organic compounds (such asglucose., acetate, etc.) as an energy or carbon source. Some algae areable to utilize either internal or external fixed carbon. A small numberof algae are obligate heterotrophs: they are incapable ofphotosynthesis, relying entirely on exogenous organic compounds asenergy and carbon sources.

Large scale cultivation of photosynthetic algae requires a relativelycontrolled environment with a large input of light energy. Therequirement for light and the high extinction coefficient of chlorophyllin these organisms has necessitated the design and development of novelsystems for cultivation and large scale growth. Chaumont, D. 1993.“Biotechnology of algal biomass production: a review of systems foroutdoor mass culture.” J. Appl. Phycol. 5:593-604. A common limitationto all of these systems is the need to supply light to the culture,making it advantageous to maximize the surface-to-volume ratio of theculture. As cell densities increase, self shading becomes a limitingfactor of productivity, resulting in relatively low biomass levels.

Most commercial production techniques use large open ponds, takingadvantage of natural sunlight, which is free. These systems have arelatively low surface area to volume ratio with corresponding low celldensities. It is also very difficult to exclude contaminating organismsin an open pond. This difficulty restricts the usefulness of open pondsto a limited number of algae that thrive in conditions not suitable forthe growth of most organisms. For example, Dunaliella salina can begrown at very high salinities. Apt K E et al, “Commercial Developmentsin Microalgal Biotechnology,” J Phycol. 35:215-226 (1999).

Enclosed photobioreactors, such as tubular photobioreactors, are analternative outdoor closed culture technology that utilize transparenttubes enclosing the culture minimizing contamination. They provide avery high surface to volume ratio, so cell densities are often muchhigher than those that can be achieved in a pond. However, even intechnologically advanced photobioreactors, the maximum algal celldensities attained are relatively low. In both ponds and bioreactors,low densities necessitate large volume cultures, which can result in asubstantial cost for harvesting the algae. Apt K E et al. (1999).Furthermore, all outdoor culture systems are subject to large variationsin light intensity and temperature caused by diurnal and seasonalperiodicity that makes maintaining maximal productivity andreproducibility problematic.

Numerous designs have also been constructed for the indoor, closedculture of algae using electric lights for illumination. Ratchford andFallowfield (1992) “Performance of a flat plate, air lift reactor forthe growth of high biomass algal cultures,” J Appl. Phycol. 4: 1-9;Wohlgeschaffen, G D et al. (1992) “Vat incubator with immersion coreillumination—a new, inexpensive set up for mass phytoplankton culture,”J Appl. Phycol. 4:25-9; Iqbal, M et al. (1993) “A flat sidedphotobioreactor for culturing microalgae,” Aquacult. Eng. 12:183-90; Leeand Palsson (1994) “High-density algal photobioreactors; usinglight-emitting diodes,” Biotechnol. Bioeng. 44:1161-7. These systems areexpensive to build and operate and are subject to the samesurface-to-volume constraints and problems associated with low densityyields as outdoor ponds. Apt K E et al. (1999).

The production costs of phototrophically grown diatoms and othermicroalgae are very expensive, resulting from low densities and highharvesting costs. Nevertheless for a small number of specific algalproducts this technology has proven very successful, producing manythousands of tons per year. Lee, Y-K (1997) “Commercial production ofmicroalgae in the Asia-Pacific Tim,” J. Appl. Phycol. 9:403-11; Apt K Eet al. (1999). Major products from photosynthetic microalgae includedried biomass or cell extracts from Chlorella, Dunaliella and Spirulina.These are primarily produced in large open ponds.

Growing algae heterotrophically in conventional fermentors is apotential alternative to ponds or photobioreactors and a potential meansto reduce substantially the cost of growing algae. Day et al. (1991)“Development of an industrial scale process for the heterotrophicproduction of a micro-algal mollusk feed,” Bioresource Technol.38:245-9; Orus et al. (1991) “Suitability of Chlorella vulgaris UAM 101for heterotrophic biomass production,” Bioresour. Technol. 38:179-184;Barclay et al. (1994) “Heterotrophic production of long-chain omega-3fatty acids utilizing algae and algae-like microorganisms,” J. Appl.Phycol. 6:123-9; Gladue and Maxey (1994) “Microalgal feeds foraquaculture,” J. Appl. Phycol. 6:131-141; Chen F., “High cell densityculture of micro algae in heterotrophic growth,” Trends Biotechnol.14:421-6 (1996); Apt, K E et al. (1999). The basic principle offermenter growth is to provide highly controlled optimal growthconditions to maximize productivity.

Typical fermenter culture conditions may be summarized as follows. Theculture vessels range in volume from 1 to 500,000 liters and areoperated under sterile conditions. A motorized shaft with a series ofimpellers provides mixing. Sterile air is pumped into the system at highpressure and flow rates to ensure proper gas exchange, and dissolved O₂and CO₂ levels are continuously monitored and adjusted. Heating and/orcooling coils regulate temperature and the automatic addition of acidand/or base maintains pH. The culture medium for algal fermentativegrowth is similar to that used for phototrophic growth, except thatglucose or a similar carbohydrate provides both fixed carbon and anenergy source in fermentative growth. Other nutrient levels (i.e.,nitrogen and phosphorus) are also continuously monitored and adjusted.Culture density may be further increased by using techniques such aschemostat culture, fed-batch culture, or membrane bioreactor culture.More detailed information regarding the growth of microalgae infermentors can be found in the papers cited herein, which are hereinincorporated by reference, and in Apt, K E et al. (1999). See, also,U.S. Pat. No. 5,244,921 to Kyle et al.; U.S. Pat. No. 5,374,657 to Kyle;U.S. Pat. No. 5,550,156 to Kyle; U.S. Pat. No. 5,567,732 to Kyle; U.S.Pat. No. 5,492,938 to Kyle et al.; U.S. Pat. No. 5,407,957 to Kyle, etal.; U.S. Pat. No. 5,397,591 to Kyle et al.; U.S. Pat. No. 5,130,242 toBarclay; U.S. Pat. No. 5,658,767 to Kyle; and U.S. Pat. No. 5,711,983 toKyle which are also incorporated by reference.

As a result of the high level of process control possible withheterotrophic growth in fermentors, culture conditions and biomassyields are consistent and reproducible, with heterotrophic algal celldensities reported of 50 grams dry biomass per liter to as high as 100 gdry biomass per liter. Gladue and Maxey (1994); Running et al. (1994)“Heterotrophic production of ascorbic acid by microalgae,” J. Appl.Phycol 6:99-104. These biomass levels are at least 10-fold higher thanthose achieved by photosynchesis-based culture systems. Radmer R J andParker B C (1994) “Commercial application of algae: opportunities andconstraints,” J. Appl. Phycol 6:93-98. The high biomass levels alsogreatly decrease the volume of water that must be processed duringharvesting per gram of biomass yield. Because cultures can routinely runin fermentors with volumes greater than 100,000 L, several thousandkilograms of dried biomass can be produced per run. The effectiveness oflarge-scale cultures and the production of high biomass levels can makethe cost of fermentative growth an order of magnitude less expensivethan photobioreactors. (Radmer and Parker 1994; Apt K E et al (1999).

The ability to provide complete control over the culture is alsocritical for maintaining food industry standard Good ManufacturingPractices (GMP), as designated by the Food and Drug Administration.Maintenance of GMP is required for the production of high-quality food-or pharmaceutical-grade materials. Apt K E et al. (1999).

The ability to grow microalgae heterotrophically using microbiologicalfermentation techniques can dramatically lower the costs associated withtheir production and provide the high degree of quality control neededfor a food grade product. The estimated cost of producing heterotrophicalgal biomass can be less than $5 per kilogram. Gladue and Maxey (1994).In contrast, the theoretical cost of producing, algae phototrophicallyin bioreactors is estimated to be an order of magnitude higher andactual production costs for phototrophic algae at aquaculture facilitiesare often two orders of magnitude higher. Wilkinson, L. (1998) “Criteriafor the design and evaluation of photobioreactors; for the production ofmicroalgae,” World Aquaculture Society Annual Meeting, February, LosVegas, Nev., p. 584; Benemann, J. R. (1992) “Microalgae aquaculturefeeds,” J. Appl. Phycol. 4:232-45. Only a small number of algae arecurrently produced using fermentation technology; these includeChlorella, Nitzschia alba, Tetraselmis, and Crypthecodinium. These algaeare able to utilize external organic compounds as energy and carbonsources.

Given the valuable products produced by algae (including algal biomassitself and the difficulties of culturing algae inphotosynthetically-based systems, it is highly desirable to culturemicroalgae in heterotrophic conditions in fermentors. However, asignificant restriction on the use of fermentation technology for theproduction of algal products is that most algae are obligate phototrophsand are therefore unable to be grown using this technology. Theretherefore exists a need to develop methods for culturing a greatervariety of algae under heterotrophic conditions in fermentors.

SUMMARY OF THE INVENTION

The present invention involves the discovery that phototrophic algae maybe transformed into heterotrophic algae, which are capable of growth inthe dark with only an external source of carbon.

It is an object of the present invention to provide methods fortransforming phototrophic algae into heterotrophic algae.

It is another object of this invention to provide such transformedalgae.

It is a further object of this invention to provide stable populationsof cells transformed according to the methods of the present invention,and to provide methods for producing biomass and other products ofphototrophic cells by culturing transformed cells in the dark.

These and other objectives are met by one or more of the followingembodiments of this invention.

This invention provides cells that are transformed with genes thatencode proteins that enhance or enable heterotrophic growth. In one modeof this embodiment, heterotrophic conversion is accomplished bytransforming the cells with only one gene. In another mode,heterotrophic conversion is accomplished by transforming the cells withmultiple genes. Cells may be transformed with a gene or genes thatencode proteins which affect uptake and catabolism of sugars and/orother exogenous sources of fixed carbon. Alternatively, cells may betransformed with a gene or genes that encode transporters capable oftaking up an exogenous fixed carbon source (i.e., a catabolyzablecompound). Or cells may be transformed with a gene or genes encodingmono- or disaccharide transporters, especially hexose transporters.

In one embodiment, the present invention provides algal cells,preferably microalgal cells, which grow in the substantial absence oflight, even though the cells are from algal strains that are obligatephototrophs, because the cells comprise chimeric DNA encoding a proteinwhich will transport a catabolizable carbon source into the algal cell.Alternatively, the present invention provides algal cells comprisingchimeric DNA which encodes a protein that will transport a catabolizablecarbon source into the algal cell, where the protein is expressed in anamount sufficient to transport into the cell adequate catabolizablecarbon source to support heterotrophic growth of the cell. Preferably,the catabolizable carbon source according to these modes of theinvention is a monosaccharide or an oligosaccharide; more preferably,the protein is a disaccharide transporter or a hexose transporter.

In another embodiment, this invention provides a method of producingalgal biomass, preferably microalgal biomass, from obligatelyphototrophic algal strains by culturing algae in the substantial absenceof light, preferably in a fermentor, using transformed cells of thealgal strain which contain chimeric nucleic acid encoding a proteinthat, upon expression by the algae, transports a catabolizable carbonsource into the algal cells. Preferably, the catabolizable carbon sourcetransported into the algal cells is a monosaccharide or anoligosaccharide; more preferably, the protein encoded by the chimericnucleic acid is a disaccharide transporter or a hexose transporter. Forthis embodiment, the protein may be expected to be expressed in anamount sufficient to transport into the cell adequate catabolizablecarbon to support heterotrophic growth of the cell.

In yet another embodiment, this invention provides a method for theheterotrophic conversion of cells of an obligately phototrophic organismselected from the group consisting of marine organisms, prokaryoticalgae, and eukaryotic algae. The method comprises the steps of (1)transforming the cells with DNA comprising a gene coding for atransporter of a catabolizable carbon source across the cell membraneand (2) selecting for cells capable of growth on the catabolizablecarbon source in the dark. In one mode of this embodiment, theobligately phototrophic organism is a marine alga. In a preferred mode,the gene coding for a transporter of a catabolizable carbon source iscoupled with a selectable gene, and after transformation, transformedcells are grown on media selective for the selectable gene beforeselecting for cells capable of growth on the catabolizable carbon sourcein the dark. In a particularly preferred mode, the selectable geneconfers resistance to an antibiotic on the transformed cells, and theselective media contains the antibiotic.

In still another embodiment, this invention provides a method forselecting transformed cells from a cell population exposed totransforming vectors containing a gene of interest. The method comprisestransfecting cells of a cell population which is unable to grow in thedark on a source of catabolizable carbon, where the transformationvector comprises a gene of interest in conjunction with a gene whoseexpression enables growth of the cells on the source of catabolizablecarbon in the dark. After the transfection, selection for cells capableof growth in the dark is carried out, and then the selected cells arefurther tested to determine whether they also contain the gene ofinterest.

In other embodiments of this invention, cells are transformed with agene or genes that encode proteins that upregulate an existingtransporter of a reduced carbon source (i.e., a catabolyzable compound)across the cell membrane. In other embodiments of this invention, cellsare transformed with a gene or genes that encode proteins that activatean existing transporter of a reduced carbon source across the cellmembrane. In still other embodiments of this invention, cells aretransformed with a gene or genes that encode proteins that facilitatethe use of reduced carbon source(s) by the cell.

In another embodiment according, to the present invention, autotrophiccells are transformed with a gene of interest in conjunction with a geneor genes encoding protein(s) that enable or enhance heterotrophicgrowth, and transformation to establish or enhance heterotrophic growthis used as a marker for transformation with the gene of interest. In yetanother embodiment of the present invention, heterotrophic cells aremutagenized to render them incapable of growth on a given carbon source,then transformed with a gene of interest in conjunction with a gene orgenes which re-establishment of the ability to grow on that carbonsource is used as a mechanism for selection, and the re-establishment ofgrowth on the carbon source is used as a marker for transformation withthe gene of interest.

A significant restriction on the use of fermentation technology for theproduction for the production of algal products is that most algae areobligate phototrophs and are therefore unable to be grown using thistechnology. It has been discovered that obligate phototrophic organismscannot utilize a fixed carbon source because they do not have themachinery to import the fixed carbon and/or they cannot convert it to ametabolically useful form. The present invention allows circumvention ofthis restriction by converting phototrophs to cells that are capable ofgrowth on external organic compounds. In order to grow in the dark on anorganic substance as a carbon and energy source, a cell must effectivelytake up the required substance(s) from the surrounding medium and thenassimilate it into all components essential for growth vialight-independent metabolic reactions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Map of the Phaeodactylum transformation vector pPha-T1 withindicated restriction sites. The sequence of this vector has the GenbankAccession No. AF219942. The fcpA promoter has been placed in front ofthe multiple cloning site (MCS). The fcpB promoter was placed in frontof the sh ble gene. The construct also contains the ampicillinresistance gene (Amp) and the E. coli origin of replication. The fcpAterminator region which follows both the MCS and the sh ble regions, isnot shown.

FIG. 2. Southern blot analyses of untransformed P. tricornutum: lanesshow results for Wild Type and two cell lines transformed with genesencoding glucose transporters. Total DNA was digested with restrictionendonuclease. A) Hybridization of DNA from cell line Hup 2 with a DNAprobe for the hup coding region. B) Hybridization of DNA from cell lineGlut 13 with a DNA probe for the glut coding region.

FIG. 3. Northern blot analyses of untransformed P. tricornutum: lanesshow results for Wild Type and two cell lines transformed with genesencoding glucose transporters. A) Hybridization of total RNA from cellline Glut 13 with a DNA probe for the glut coding region. B)Hybridization of total RNA from cell line Hup 2 with a DNA probe for thehup coding region.

FIG. 4. Reactivity of antibodies specific for Glut1 or GFP to membraneproteins extracted from untransformed and transformed cell lines. Theproteins were resolved by SDS-PAGE following solubilization of totalmembranes from Wt, untransformed cells; G1-40, the Glut1GFP-40transformant; B, human erythrocytes; Glut1-17, the Glut1-17transformant. The antibodies used were specific for GFP (left panel) orGlut1 (right panel).

FIG. 5. Uptake of glucose by transformed and untransformed cell lines.Both Glut1GFP-40 and Glut1-17 were assayed for glucose uptake using theprocedure described herein.

FIG. 6. Localization of Glut1 in P. tricornutum transformed with thechimeric gene encoding the Glut1-GFP fusion protein. The top panel showsa transmitted light image of untransformed P. tricornutum cells (A),fluorescence from the cells in the red channel (B), which representschlorophyll fluorescence, and fluorescence from the cells in the greenchannel (C). The lower panel shows fluorescence in the green channel ofcells that have been transformed with the GFP gene driven by an Feppromoter (D), and of cells that have been transformed with the chimericGlut1-GFP gene (E, strain designated Glut1-40). All fluorescence imagesare single confocal optical sections. Scale bars=10 microns.

FIG. 7. Growth of wild-type cells and Glut1-17 under differentconditions. The left panel shows the growth of wild type cells (Wt) andthe right panel shows growth of Glut1-17 under various conditions. Cellswere cultured in the light without supplemented glucose (closedcircles), in the dark with supplemented glucose (closed triangles) or inthe light with supplemented glucose (open circles).

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

There are a number of valuable commercial uses for microalgae. However,commercial-scale production of photosynthetic algae most frequentlyinvolves the use of large, outdoor ponds, which present numerousdisadvantages. Contaminants frequently invade pond cultures since theyremain unshielded from the environment, and seasonal variations andfluctuations in temperature and light conditions make it difficult topredict the rate of growth and final culture densities. Furthermore,self-shading among the algae can severely limit biomass yields. Thesefactors have restricted successful large-scale cultivation of algae to asmall subset of organisms that includes Spirulina and Dunaliella (Apt,et al. (1999) J. Phytol, 35:215).

A strategy that has the potential to dramatically reduce the cost ofproducing macroalgal biomass for commercial uses is to engineer theorganisms to grow without light in conventional microbial fermentors.Considerations discussed above suggest that it would be advantageous toengineer micro algal metabolism for high rates of heterotrophic growth.Most photosynthetic organisms (plants, algae, cyanobacteria) are capableof gluconeogenesis for the storage of carbon as sugars. When the carbonsupply is limited, these reserves are metabolized for energy usingfamiliar glycolytic mechanisms. The present inventors explored whetherphotosynthesis could be obviated if a cell could directly import fixedcarbon for glycolysis, as with any natural heterotroph.

Most diatoms do not have the capacity to grow in the absence of light onexogenous glucose (Droop, 1974). A ‘metabolic block’ that preventsheterotrophic growth of diatoms was postulated to result from theinability of the cells to take up or further metabolize sugars. Thepresent inventors have demonstrate herein that trophic conversion of theobligate photo autotrophic diatom, P. tricornutum, can be achieved bytransforming the alga with a single gene encoding a glucose transporter.Strains transformed with either the Hup1 gene of C. kessleri or theGlut1 gene of humans are able to take up glucose and grow in the darkwith glucose as the sole source of reduced carbon. These results clearlydemonstrate an engineered trophic conversion of an obligatephotoautotrophic organism into a heterotrophic organism. While Hup1 hasalso been expressed in the green alga Volvox (Hallman, et al., 1996) andthe diatom Cylindrotheca (Fischer, et al., 1999), neither of thesetransformed strains were able to grow heterotrophically.

Conversion of a phototrophic alga to one capable of growth on, e.g.,glucose as the only carbon source requires the introduction of genesencoding the required functions. In order for an obligate phototroph tobe converted to a heterotroph, the phototroph must acquire competencefor growth when supplied with an exogenous source of fixed carbon. Thus,conversion of a phototrophic alga to one capable of growth in the darkon an external organic substance, such as glucose, as the sole carbonand energy source requires the introduction of a gene or genes encodingthe required functions (e.g., transporters, hexokinase, etc.).

Previous attempts to convert obligate phototrophic algae intoheterotrophic algae have failed. Although algae transformed to expresssugar transporters have been shown to be capable of taking up glucose,none of these algae have been able to grow in the dark.

The present invention provides a method for converting obligatephototrophic cells into heterotrophic cells, which are capable of growthin the dark. There is no obvious precedent for this type of conversion,so it has been difficult to predict what types of complications will beencountered. According to the present invention, at the simplest level,only a hexose transporter is required for the conversion. The discoveryby the present inventors that heterotrophic conversion can beaccomplished by the insertion of a single gene (e.g., a gene encoding atransporter capable of taking up an exogenous fixed carbon source, suchas a gene encoding a hexose transporter) is surprising considering thecomplexity of heterotrophic metabolism and the previous failures in theart.

Of course, because heterotrophic growth is a complex process,heterotrophic growth may be enhanced by introduction of multiple genesthat code for proteins that enhance or enable the uptake and/orcatabolism of sugars and/or other exogenous sources of fixed carbon. Inthe present invention, phototrophic algae is transformed with one ormore genes affecting uptake and catabolism of sugars and/or exogenoussource of fixed carbon. The cells are transformed with these genes usinga suitable transformation vector. Preferably, the gene(s) inserted underthe control of a promoter will result in expression of the gene(s) inthe transformed cells.

DEFINITIONS

In describing the present invention, the following terminology is usedin accordance with the definitions set out below.

“Heterotrophic conversion” is the conversion of a phototrophic,autotrophic, or auxotrophic organism that is incapable of growth on, orwhich grows poorly on, a given organic carbon and energy source (e.g.,glucose), to an organism capable of heterotrophic growth, or improvedheterotrophic growth, on that same carbon source. For cells to beheterotrophically converted, it is necessary that they be capable ofgrowth in the dark. It is not required that the cells remain in constantor complete darkness.

“Catabolism” is metabolic breakdown or degradation of a complex molecule(i.e. glucose, or other carbon source) into simplier products, primarilyfor the production of energy. A “catabolizable carbon source” is acomplex molecule, typically a mono- or oligo-saaccharide, an amino acid,or other biochemical molecule, that can undergo catabolism in abiological cell.

“Improved growth” is intended to encompass any improvement. Asnon-limiting examples, improvement could comprise more rapid growth,more rapid production of a desired product, or growth which may besustained for longer periods of time. Likewise, poor growth” is intendedto encompass any growth-related feature which is sought to be improved.Both “poor growth” and “improved growth” are relative terms andquantitative levels denoting poor or improved for one characteristic ororganism may not be poor or improved for another characteristic ororganism.

A “protein which can enable or enhance heterotrophic growth” is meant tobe interpreted broadly to encompass any protein that (1) makes itpossible or assists in making it possible for a cell which was incapableof growth on a given carbon source to grow on that carbon source, (2)makes it possible or assists in making it possible for a cell which grewpoorly on a given carbon source to exhibit improved growth on thatcarbon source, or (3) makes it possible or assists in making itpossible—for a cell which was initially capable of growth on a givencarbon source to exhibit improved growth on that carbon source. Suchproteins include, but are not limited to, transporters of carboncompounds across the cell membrane, proteins which catabolite carboncompounds, and proteins which upregulate and/or activate suchtransporters or catabolizing proteins.

A “phototroph” is an organism capable of converting light energy tochemical energy.

An “autotroph” is an organism cells that uses inorganic material as asource of nutrients and CO₂ as its source of carbon.

An “obligate phototroph” is an organism that requires light energy forthe production of chemical energy and is incapable of using exogenoulysupplied performed organic molecules as its sole source of carbon orenergy.

A “heterotroph” is an organism that can use preformed organic compoundsas the source of carbon and energy in the absence of light. Heterotrophscan, therefore, grow independently of illumination; for example,heterotrophs can grow in the dark, in the light, and in partial light.Similarly, “heterotrophic growth” refers to growth which does notrequire light to occur and can, therefore, occur independent of thelevel or lack of illumination.

“Auxotrophs” are organisms which require a certain substance to grow.For example, a given auxotroph may be unable to produce a specific aminoacid and will therefore require that amino acid for growth. Manyauxotrophs are phototrophs. According to the present invention,heterotrophic conversion includes the conversion of an organism that isincapable of growth (or which grows poorly) on an external carbon sourcesuch as glucose to an organism capable of growth (or which grows well)on that external carbon source.

“Phototroph,” “autotroph,” and “auxotroph” may all be usedinterchangeably to indicate an organism that does not grow, or thatgrows poorly, on the external carbon source, e.g., glucose, on which itis sought to be grown. Because these organism grow poorly or do not growat all on external carbon sources, they may also be referred to asobligate photo auto-, or auxotrophs in the context of this invention.

As used herein, “growth (or culture) in substantial darkness (orsubstantial absence of light),” “grown (or cultured) in substantialdarkness (or substantial absence of light),” and like phrases indicatethat the growing or culturing is carried out under light conditionsunder which phototrophic cells would be unable to grow or would growvery poorly. Similarly, “substantial darkness” and “substantial absenceof light” are synonymous and indicate a level of illumination which mayvary between total darkness and the level at which phototrophic cellscannot grow or grow very poorly. Expressed in an alternative manner, thelevel of illumination indicated by the phrases “in the dark,”“substantial absence of light,” and like phrases is a level ofillumination which would be growth-limiting (in terms of, asnon-limiting examples, doubling time, maximum culture density, orproduct production) for phototrophic cells. Hence, growth or culture inthe dark encompasses, as non-limiting examples, growth in fermentorshaving windows or openings for observing the cells, feeding the cells,and the like, so long as light entering the fermentor is insufficient tosupport long-term growth of obligate phototrophs in culture.

A “chimeric DNA” is an identifiable segment of DNA within a larger DNAmolecule that is not found in association with the larger molecule innature. Thus, when the chimeric DNA encodes a protein segment, thesegment coding sequence will be flanked by DNA that does not flank thecoding sequence in any naturally occurring genome. Allelic variations ornaturally occurring mutational events do not give rise to a chimeric DNAas defined herein.

A “coding sequence” is an in-frame sequence of codons that (in view ofthe genetic code) correspond to or encode a protein or peptide sequence.Two coding sequences correspond to each other if the sequences or theircomplementary sequences encode the same amino acid sequences. A codingsequence in association with appropriate regulatory sequences may betranscribed and translated into a polypeptide in vivo. A polyadenylationsignal and transcription termination sequence will usually be located 3′to the coding sequence.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream codingsequence. A coding sequence is “under the control” of the promotersequence when RNA polymerase which binds the promoter sequence willtranscribe the coding sequence into mRNA, which is then in turntranslated into the protein encoded by the coding sequence. For purposesof defining the present invention, the promoter sequence is bounded atits 3′ terminus by the translation start codon of a coding sequence andextends upstream (5′ direction) to include at least the minimum numberof bases or elements necessary to initiate transcription at levelsdetectable above background. Within the promoter sequence will be founda transcription initiation site (conveniently defined by mapping withnuclease S1), as well as protein binding domains (consensus sequences)responsible for the binding of RNA polymerase. Promoters will usuallycontain additional consensus sequences (promoter elements) for moreefficient initiation and transcription of downstream genes.

A “genetic fusion” according to this invention is a chimeric DNAcontaining a promoter and a coding sequence that are not associated innature.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

Vectors are used to introduce a foreign substance, such as DNA, RNA orprotein, into an organism. A “DNA vector” is a replicon, such asplasmid, phage or cosmid, to which another DNA segment may be attachedso as to bring about the replication of the attached segment.

A cell has been “transformed” by exogenous DNA when such exogenous DNAhas been introduced inside the cell wall. Exogenous DNA may or may notbe integrated (covalently linked) to chromosomal DNA making up thegenome of the cell. For example, the exogenous DNA may be maintained onan extrachromosomal element (or replicon) such as a plasmid. A stablytransformed cell is one in which the exogenous DNA has become integratedinto a chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe cell to establish cell lines or clones comprised entirely of apopulation of daughter cells containing the exogenous DNA.

“Salt” as used in herein refers to an inorganic ionic compound which iscommonly found in sea water or a mixture of such compounds. Theconcentration of salt is expressed herein as the amount of suchcompounds that provide the ionic equivalent to a given weight of sodiumchloride. The salt concentration of seawater is about 32-33 g/L.

Algae which are obligate phototrophs, (both macro- and micro-algae) maybe converted to heterotrophs by the method of this invention. Many algaeare obligate phototrophs, which are incapable of growth on glucose, andany of these organisms are potential targets for trophic conversion.Lists of phototrophs may be found in a review by Droop (Droop M R“Heterotrophy of Carbon.” In Algal Physiology and Biochemistry,Botanical Monographs, 10: 530-559, ed. Stewart W D P, University ofCalifornia Press, Berkeley (1974)); and a representative, non-exclusivelist of phototrophic algal genera with potential or known commercialvalue is provided below (Table A), grouped at the phylum level. The“common name” is in parenthesis.

TABLE A Exemplary List of Phototrophic Algae Cyanophyta (Blue-greenalgae) - Spirulina, Anabaena. Chlorophyta (Green algae) - Dunaliella,Chlamydomonas, Heamatococcus. Rhodophyta (Red algae) - Porphyridium,Porphyra, Euchema, Graciliaria. Phaeophyta (Brown algae) - Macrocystis,Laminaria, Undaria, Fucus. Baccilariophyta (Diatoms) - Nitzschia,Navicula, Thalassiosira, Phaeodactylum. (Some members of these generaare capable of heterotrophic growth on glucose, but not others.)Dinophyta (Dinoflagellates) - Gonyaulax. Chrysophyta (Golden algae) -Irsochrysis, Nannochloropsis Cryptophyla - Crypromonas. Euglenophyta -Euglena.

Phaeodactylum tricornutum has been one of the most widely utilized modelsystems for studying diatoms, particularly in areas of ecology,physiology, and biochemistry. See, Ianora A, Poulet S A, Miralto (1995),“A comparative study on the inhibitory effect of diatoms on thereproductive biology of the copepod Temora stylifera,” Mar. Biol.121:533-539; Kuma K, Matsunga K (1995); “Availability of colloidalferric oxides to coastal marine phytoplankton,” Marine Biol. 122:1-11;Alwyn T, Rees V (1995), “On ammonia futile cycling in a marineunicellular alga,” Biochem. Biophys. Acta. 1228:254-260; La Roche I etal. (1995), “Flavodoxin expression as an indicator of iron limitation inmarine diatoms,” J. Phycol. 31:520-530; Rees V et al. (1995), “In situglutamine synthetase activity in a marine unicellular alga. Developmentof a sensitive colorimetric assay and the effects of nitrogen status onenzyme activity,” Plant Physiol. 109:1405-1410; Khalyfa A et al. (1995)“Purification and characterization of chlorophyllase from the algaPhaeodacrylum tricornitum by preparative native electrophoresis,” Appl.Biochem. Biotech 53:11-27; Zhukova N N, Alzdaischer N A (1995), “Fattyacid composition of 15 species of marine microalgae,” Phytochemistry39:351-356. Recently it has also been employed as a molecular model tostudy some of the unique biological processes found in diatoms,particularly involving plastid protein targeting and cell wallformation. Apt K E, Clendennen S K, Powers D A, Grossman A R (1995) “Thegene family encoding the fucoxanthin chlorophyll proteins from the brownalga Macrocystis pyrifera,” Mol. Gen. Genent. 246:455-64; Apt K E et al.(1994), “Characterization of genes encoding the light-harvestingproteins in diatoms: biogenesis of the fucoxanthin chlorophyll a/cprotein complex,” J Appl. Phycol, 6:225-230, Bhaya D, Grossman A G(1993) “Characterization of gene clusters encoding the fucoxanthinchlorophyll protein of the diatom Phaeodactylum tricornutum,” NucleicAcids Research 21:4458-68):

The microalga Phaeodactylum tricornutum has been repeatedly reported tobe unable to utilize external glucose, acetate, amino acids, etc, as thesole energy or carbon source. Cooksey K E “Acetate metabolism by wholecells of Phaeodactylum tricornutum,” J. Phycol. 10:253-7 (1974); Droop1974; Hellebust J A, Lewin J “Heterotrophic Nutrition,” in (Werner Ded.) The Biology of Diatoms, Botanical Monographs 13:169-197, Universityof California Press, (1977). The present inventors have repeatedlyexamined this microalga and observed no detectable growth or nutrientuptake in the dark on media containing glucose. The recent developmentof a genetic transformation system has provided new opportunities tostudy the molecular mechanisms for the biological processes in organismssuch as Phaeodactylum. (WO 97/39106 and U.S. Pat. No. 6,027,900 toAllnutt et. al., both entitled: “Methods and Tools for Transformation ofEukaryotic Algae,” both incorporated herein by reference). As a result,Phaeodactylum tricornutum was selected as a model to test the ability toconvert an obligate phototrophic organisms through the use of geneticengineering.

Transformed cells are produced by introducing exogenous DNA into apopulation of target cells and selecting the cells which have taken upthe exogenous DNA, usually by measuring expression of some determinantpresent on the exogenous DNA but missing from the non-transformed cells.A preferred selective marker for use in algae is the Zeocin (Invitrogen)resistance selection system described in WO 97/39106. See, also, U.S.Pat. No. 6,027,900 to Allnutt et al. Briefly, resistance to zeocin (andrelated antibiotics) has been discovered to be maintained in high saltmedium to a much greater extent than is observed for other antibioticresistance genes. Thus, transformants containing exogenous DNA with azeocin resistance determinant will grow in salt water in the presence ofsuitable concentrations of zeocin, while non-transformed cells of marineorganisms will not.

Standard methods for construction of chimeric DNA and its use intransformation of cells and expression of proteins therein are familiarto the skilled worker, and these methods have been described in a numberof texts for standard molecular biological manipulation. (See, e.g.,Packer & Glaser, 1988, “Cyanobacteria”, Meth. Enzymol., Vol. 167;Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology,Academic Press, New York; Sambrook, J., Maniatis, T., & Fritsch, E. F.(1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y.; and Clark M S, 1997, PlantMolecular Biology, Springer, N.Y.

The chimeric nucleic acid of the present invention, e.g., a nucleic acidconstruct comprising a coding sequence which encodes a protein which canenable or enhance heterotrophic growth fused to a suitable promoter, maybe introduced into cells of, e.g., marine organisms or eukaryotic algaealone or as part of a DNA vector. Suitable vectors for use in marineorganisms or eukaryotic algae are known in the art and any such vectormay be used. Even vectors which do not replicate in algae can be useful,if recombination between the vector and the genome occurs.

To summarize the process of constructing a vector, the upstream DNAsequences of a gene expressed under control of a suitable promoter maybe restriction mapped and areas important for the expression of theprotein characterized. The exact location of the start codon of the geneis determined and, making use of this information and the restrictionmap, a vector may be designed for expression of a heterologous proteinby removing, the region responsible for encoding the gene's protein butleaving the upstream region found to contain the genetic materialresponsible for control of the gene's expression. A syntheticoligonucleotide multicloning site is preferably inserted in the locationwhere the protein coding sequence once was, such that any gene (e.g., aprotein coding sequence) could be cloned in using restrictionendonuclease sites in the synthetic oligonucleotide. An unrelated gene(or coding, sequence) inserted at this site would then be under thecontrol of an extant start codon and upstream regulatory region thatwill drive expression of the foreign (i.e., not normally associated withthe regulatory sequences of the vector) protein encoded by this gene.Once the gene for the foreign protein is put into a cloning vector, itcan be introduced into the host organism using any of several methods,some of which might be peculiar to the host organism.

DNA delivery methods which are within the skill of the art includesilica carbide whisker vortexing, glass bead vortexing, electroporation,and particle bombardment. Preferably, glass bead vortexing is used. Morepreferably, particle bombardment is used. These methods may be used tointroduce the genetic fusion of the present invention into cells of,e.g., marine organisms or eukaryotic algae alone or as part of a DNAvector.

Variations on these methods are amply described in the generalliterature references cited herein. Manipulation of conditions tooptimize transformation for a particular host is within the skill of theart.

Suitable transformation vectors include those having at least one sitefor inserting a gene coding for a protein that enhances or enablesheterotrophic growth. Preferably, transformation vectors used in themethods according to the present invention have at least an insertionsite for insertion of a gene coding for a transporter capable of takingup an exogenous fixed carbon source. More preferably, transformationvectors used in the methods according to the present invention have atleast an insertion site for insertion of a gene encoding a hexosetransporter. Typically, transformation vectors are also able toreplicate in bacteria, so that large amounts of the vector can beprepared easily, using methods such as those described in Sambrook, I.,Maniatis, T., & Fritsch, E. F. (1989) Molecular Cloning: A LaboratoryManual. 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.Preferably, vectors will be chosen which are known to transfect the hostalgal species.

Transformation systems have been described for a limited number ofeukaryotic algal genera, including diatoms. For example, four species ofdiatoms have been transformed. In each case a special vector wasdeveloped for the diatom of interest. The present inventors transformedPhaeodactylum, using the vector called pPha-T1, which will be describedin more detail below. Cyctotella cryptica and Navicula saprophila havebeen transformed using vectors pACCNPT10 and pACCCNPT5.1, which have thenptII gene (neomycin phosphotransferase) conferring resistance to theantibiotic G418. The promoter driving expression of introduced gene wasfrom the endogenous acc gene (acyl-CoA carboxylase). Dunahay, T. G.,Jarvis, E. E. & Roessler, P. G. (1995) “Genetic transformation of thediatoms Cyclotella cryptica and Navicula saprophila,” J. Phycol.31:1004-12. Cylindrotheca fusiformis has been transformed by Fisher etal. (Fischer H., Robl, I., Sumper, M., & Kroger, N., (1999), “Targetingand covalent modification of cell wall and membrane proteinsheterologously expressed in the diatom Cylindrotheca fusiformis(Bacillariophyceae),” J. Phycol. 35:113-20). In this case, the sh blegene, which confers resistance to the antibiotic zeocin was utilized.The promoter driving expression was from the endogenous fru gene, whichencodes for a cell wall protein, and the vector was called pble/fruE Aderivative of this vector, pble/HUPtag, containing the hup1 geneencoding the glucose transporter from Chlorella, was also used totransform Cylindrotheca fusiformis. The transformed alga was able totake up glucose. However, the transformed alga was unable to growheterotrophically (Fischer, et al., 1999).

A small number of green algae have been transformed (see Stevens andPurton (1997) “Genetic engineering of eukaryotic algae: progress andprospects,” J. Phycol. 33:713-22). The most prominent is Chlamydomonas.This algae has been a significant molecular model system for many yearswith numerous labs involved in research. As a result there are a largenumber of described vectors that could be used for trophic conversion ofthis alga. Volvox, a genus that is closely related to Chlamydomonas, hasalso been transformed (Hallman A, Sumper M (1996) “The Chlorellahexose/H symporter is a useful selectable marker and biochemical reagentwhen expressed in Volvox,” Proc. Natl. Acad. Sci. 93:669-673). Hallmannand Sumper introduced the Chlorella hup gene into Volvox. The alga wassensitive to deoxyglucose, indicating the transporter was functioning.However, the authors did not provide uptake measurements and the cellsdid not grow heterotrophically.

Preferred vectors for this invention are vectors developed for one ormore algal systems. Furthermore, the methods described herein and theZeocin resistance selection system provide one skilled in the art withguidance for the preparation and selection of vectors appropriate fortransformation of phototrophic algae into heterotrophic algae.

Different promoters may be more or less effective in different hostsystems. For example, the promoter from a gene associated withphotosynthesis in one photosynthetic species may be used to directexpression of a protein in transformed algal cells or cells of anotherphotosynthetic marine organism. Suitable promoters for use in thepresent invention may be isolated from or synthesized based on knownsequences from other photosynthetic organisms. Preferred promotersinclude those for genes from other photosynthetic species, includingother algae and cyanobacteria, which are homologous to thephotosynthetic genes of the algal host to be transformed. Preferably,the promoters chosen for use according to the present invention providefor constitutive, high level production of the chimeric genes theycontrol in the organisms in which they are used. Preferably, thisconstitutive, high level production is independent of illumination, sothat it occurs both in light and in the absence of light. Examples ofsuch promoters are the promoters for constitutively expressed genes suchas housekeeping genes. Given the reporter gene systems now available,selection of a promoter with the desired characteristics is well withinthe ability of one in the arts of molecular biology, phycology, and thelike. See, Zaslayskaia, et al.

For a particular algal host, it may be preferable to identify thehomologous light harvesting gene from that host and isolate thehomologous promoter from the host organism to get optimal expression.However, it has been found for Some organisms that foreign promotersgive excellent or less restricted expression in the host relative to thehomologous promoter of the host organism (Mermet-Bovier, et al. (1993)Curr. Microbiol. 26:323-327).

In one embodiment, the gene(s) desired to be expressed is regulated by apromoter of a chlorophyll binding protein. A series of light harvestingpromoters from the fucoxanthin chlorophyll binding proteins have nowbeen identified and cloned from Phaeodactylum tricornutum by the presentinventors. See, Apt, et al. (1996). The use of fcp promoters fortransformation of algae, especially photosynthetic diatoms, is describedin WO 97/39106. See, also, U.S. Pat. No. 6,027,900 to Allnutt et al.Suitable promoters include the fcpA, fcpB, fcpC, and fcpE promoters, aswell as many other the (light harvesting complex) promoters. Othersuitable promoters which are apparent to the skilled worker may be usedfor heterotrophic conversion according to this invention.

A general-purpose transformation vector, pPha-T1 (FIG. 1) has beenconstructed to facilitate efficient introduction of heterologous genesinto the diatom P. tricornutum. This vector contains a multiple cloningsite with ten unique restriction sites for inserting genes of interest.The promoter and terminator regions of the fcpA gene flank the multiplecloning site to promote efficient expression of the inserted genes. Theprimary selection for P. tricornutum cells harboring the vector iszeocin resistance, which is encoded by the sh ble gene flanked by the P.tricornutum fcpB promoter and the fcpA terminator.

The plasmid transformation vector pPha-T1 (FIG. 1) was constructed inseveral stages. The first step involved subcloning the fcpA terminatorregion from pfcpA/ble (Apt, et al., 1996) into the HinDIII-Xhol sites ofpSP73 (Promega). The zeocin resistance cassette from pfcpB/ble (Apt etal., 1996), which contains the fcpB promoter driving the sh ble gene,was subcloned as a XhoI fragment into the pSP73 XhoI site. The fcpApromoter region from pfcpA/ble (Apt et al., 1996) was subcloned as aPstI-EcoRI fragment and inserted into Bluescript SK−. This same fragmentwas then removed as a BamHI-EcoRV fragment and ligated into theBgIII-EcoPV sites of pSP73 to form the final basic construct. Themulti-cloning site from pSP73 between the EcoRV and HinDIII sites waspreserved intact, excluding the Clai site, which was removed bysite-directed mutagenesis to eliminate a cryptic ATG start codon (Deng,W. P. & Nickotoff, J. A., (1992) “Site-directed mutagenesis of virtuallyany plasmid by eliminating a unique site,” Anal, Biochem. ‘1100:81-8;Zaslayskaia, et al., 2000).

Plasmid transformation vector pPha-T1 may be used to transform diatomssuch as Cyclotella, Cylindrotheca, and Nitzschia alba by particlebombardment. The skilled worker may also use alternative transformationvectors, based on homologous promoters, which can be prepared by methodsused to prepare the pPha-T1 vector. WO 97/39106. See, also, U.S. Pat.No. 6,027,900 to Allnutt et al.

The ability of a given promoter to regulate gene expression, as well asthe timing of such expression and factors affecting such expression, inany particular species of algae can be evaluated using the Zeocinselection system described in WO 97/39106. See, also, U.S. Pat. No.6,027,900 to Allnutt et al. Additional selection systems which can beused to evaluate promoters and their activity in a given organism aredescribed in Zaslayskaia, et al., 2000).

In order to easily detect the transfer of genetic material to anyorganism, i.e., in order to detect whether a cell has been transformed,phenotypic trials indicating this transfer need to be established. Thetrait most conveniently, and therefore traditionally, manipulated hasbeen antibiotic sensitivity. The lowest concentrations of zeocin andnourseothricin which completely abrogate growth in various organisms maybe determined using the methods elaborated for P. tricornutum in WO97/39106. See, also, U.S. Pat. No. 6,027,900 to Allnutt et al., andExample 1, below. Additional selection systems which can be used todetect the transfer of genetic material to an organism are described inZaslayskaia, et al. (2000).

Suitable coding sequences for use in the genetic constructs of thepresent invention include riles for proteins that enhance or enableheterotrophic growth. Preferably, coding sequences are genes that encodeproteins which affect uptake and catabolism of sugars and/or otherexogenous sources of fixed carbon. More preferably, coding sequences aregenes that encode transporters capable of taking up an exogenous fixedcarbon source. Non-limiting examples of such carbon sources includesugars, fatty acids, amino acids, pyruvate, glycerol, and citrate. Evenmore preferably, coding sequences are genes encoding mono- ordisaccharide transporters, such as, but not limited to, sucrosetransporters. Most preferably, coding sequences are genes encodinghexose transporters, such as, but not limited to, glucose transporters.

Other non-exclusive examples of coding sequences which are suitable forin the genetic constructs of the present invention include codingsequences that encode proteins that upregulate existing transporters ofreduced carbon sources across the cell membrane, coding sequences thatencode proteins that activate existing transporters of reduced carbonsources across the cell membrane, and coding sequences that encodeproteins that facilitate the catabolism of reduced carbon sources by thecell.

The inventors have discovered that expression of a single exogenous geneencoding a transporter for a compound that serves as a catabolyzablecarbon source may be sufficient to convert an obligate phototroph to arecombinant cell capable of heterotrophic growth. In preferredembodiments of this invention, transfection with a single gene effectsthe phototroph-to-heterotroph conversion. Of course, introduction of aplurality of genes may be necessary or desirable to achieve more ampleheterotrophic growth, and constructs using combinations of suitablecoding sequences may be used according to this invention. For example,sequences encoding transporters for different compounds or transportersin conjunction with enzymes involved with metabolism of the compoundsmay be used.

Hexose transporters comprise a superfamily of related sugar transportersthat all consist of a structure having 12 membrane-spanning domains.See, e.g., Walmsley A R et al. (1998) “Sugar transporters from bacteria,parasites and mammals: structure-activity relationships,” Trends.Biochem. Sci. 23(12):476-81 (a broad general review); Henderson P J,(1990) “The homologous glucose transport proteins of prokaryotes andeukaryotes,” Res. Microbiol. 141(3):316-28; Bisson L F et al., (1993)“Yeast sugar transporters,” Crit. Rev. Biochem. Mol. Biol.28(4):259-308; Kruckeberg (1996); Mueckler M et al., (1997); Barrett M Pet al., (1998) “Trypanosome glucose transporters,” Mol. Biochem.Parasitol. 91(1):195-205; Caspari T et al. (1994) “Hexose/H+ symportersin lower and higher plants,” J. Exp. Biol. 196; 483-491. An exemplarylist of characterized hexose transporters is given in Table B, alongwith examples of characterized sucrose, citrate, and fatty acidtransporters. See, Hirsch D. et al., (1998) “A family of fatty acidtransporters conserved from mycobacterium to man,” Proc. Natl. Acad.Sci. U.S.A. 95(15): 8625-8629; Heisterkamp N. et al., (1995)“Localization of the human mitochondrial citirate transporter proteingene to chromosome 22Q11 in the DiGeorge syndrome critical region,”Genomics 29(2): 451-456; Rentsch D et al., (1998) “Structure andfunction of plasma membrane amino acid, oligopeptide and sucrosetransporters from higher plants,” J. Membr. Biol. 162(3):177-90. Table Balso includes an exemplary list of amino acid transporters. See, Saier MH, (2000) “Families of transmembrane transporters selective for aminoacids and their derivatives.”Microbiology 146:1775-95; Meredith D, andBoyd C A. (2000) Structure and function of eukaryotic peptidetransporters. Cell Mol Life Sci 57(5):754-78; Ortiz-Lopez A, Chang H,and Bush D R. (2000) “Amino acid transporters in plants.” BiochimBiophys Acta 1465(1-2):275-80; and McGivan J D. (1996) “Mammalian aminoacid transporters and their regulation: introduction.” Biochem SocTrans. 24(3):837-8.

TABLE B Examples of characterized transporters of reduced carbon sources(i.e., catabolyzable compounds) Examples of characterized hexosetransporters Yeast - hxt 1-7, gh rl Mammals - glut1, glut4Trypanosomes - tht Plants - stpl-4 Algae - hupl, hup2 Cyanobactria -glcP Bacteria - xylE, galP Examples of characterized sucrose(disaccharide) transporters Plant - sutl, sut2, sucl Examples ofcharacterized citrate transporters Mammalian - ctp Bacteria - citExamples of characterized fatty acid transporters Mammalian - fatpYeast - , pxalp, fatlp Examples of amino acid transporters Plant -aap1-5 Mammalian - ngt1 yeast - ort1 bacteria - gap1

Since the different sugar transporters are related, any of them may beused as a transporter in trophic conversion according to the presentinvention. The present inventors have successfully convertedphototrophic algae to heterotrophs by inserting glut1 (mammalian hexosesymporter) or hup1 (algal hexose symporter). The present inventors havediscovered that insertion of glut1 is particularly successful inconverting phototrophic algae into heterotrophs. Experiments with hxt1,hxr2, hxt4 (yeast hexose symporters) were less successful. The lessersuccess experienced when transforming Phaeodactylum with yeasttransporter genes is most likely the result of mismatched codon usage.For example, the yeast transporters use a codon bias which does notmatch the Phaeodactylum codon usage.

Many algae have been shown to have unusual codon usages (Bhaya D.,Grossman A G (1993) “Characterization of gene clusters encoding thefucoxanthin chlorophyll protein of the diatom Phaeodactylumtricornutum,” Nucleic Acids Research 21:4458-159; Apt K E, Hoffman N,Grossman A R (1993) “The γ subunit of R-phycoerythrin and its possiblemode of transport into the plastid of red algae,” J Biol. Chem.268:16208-15; Apt K E, Clendennen S K, Powers D A, Grossman A R (1995)“The gene family encoding the fucoxanthin chlorophyll proteins from thebrown alga Macrocystis pyrifera,” Mol. Gen. Genent. 246:455-64). Thecodon usage in P. tricornutum is very similar to that of humans in thatthe arginine codons AGA and AGG are rarely used (Bhaya et al., 1993);Apt, et al., 1995). Previous attempts to express in P. tricornutumheterologous genes which contained a preponderance of the AGA codon allfailed. The present inventors have determined that the infrequent use ofcodons AGA and, especially, AGG, are important for appropriate geneexpression. To date, there have not been any genes cloned from, orsuccessfully expressed in P. tricornutum that contain a predominance ofthe codon AGG. According to the present invention, all P. tricornutumtransformations are therefore preferably done with genes that do notcontain these codons (or at least rarely contain them).

Briefly, the DNA sequence of a protein of interest is determined.Undesirable codons are identified. Using point mutagenesis, theseundesirable codons are replaced by desirable codons coding for the sameamino acid. The newly reconstructed genes are then inserted into vectorsand used to transform species of interest. In this way, heterogeneousproteins can be tailored to be efficiently expressed in organisms ofinterest. Therefore, any genes related to uptake and use of sugar and/orother exogenous sources of fixed carbon may be reconstructed by suchtechniques and used to convert phototrophs to heterotrophs using themethods of the present invention. For example, the yeast hexosetransporters (the protein products of the hxt genes) may be used moresuccessfully to convert Phaeodactylum by reconstructing the hxt genes toreduce or eliminate the use of codons AGA and AGG.

Alternatively, codon usage can be changed by reconstructing the gene’using conservative amino acid substitution (Zolotukhin S, Potter M,Hauswirth W M, Guy J, Muzyczka N (1996) “A ‘humanized’ green fluorescentprotein cDNA adapted for high-level expression in mammalian cells,” J.Virology, 70:4646-4654; Haas J, Park E C, Seed B (Mar. 1, 1996) “Codonusage limitation in the expression of HIV-1 envelope glycoprotein,”Curr. Biol. 6(3):315-24). “Conservative amino acid substitutions” arethe substitution of one amino acid residue in a sequence by anotherresidue of similar properties, such that the secondary and tertiarystructure of the resultant peptides are substantially the same.Conservative amino acid substitutions occur when an amino acid hassubstantially the same charge as the amino acid for which it issubstituted and the substitution has no significant effect on the localconformation of the protein. Amino acid pairs which may beconservatively substituted for one another are well-known to those ofordinary skill in the art.

Whether a given gene has been successfully expressed in a giventransformed cell (as determined by using the Zeomycin resistanceselection system) can be determined using the methods of the presentinvention. Successful expression of a gene or genes useful forconverting phototrophic cells to heterotrophic cells may be measured bythe ability of the transformed cells to take up an external source offixed carbon (e.g., glucose, acetate) or to grow in the dark.Preferably, successful expression of a gene or genes useful forconverting phototrophic cells to heterotrophic cells is measured by boththe ability of the transformed cells to take up an external source offixed carbon and to grow in the dark. Control cells for use indetermining the success of expression in this context can be negativecontrols (e.g., cells of the transformant species which have not beentransformed or other phototrophs) and/or positive controls (e.g.,naturally occurring heterotrophs or phototrophic cells which havepreviously been converted to heterotrophs).

A DNA sequence encoding a gene encoding a protein useful for the uptakeand/or use of an exogenous source of carbon may be inserted in thechosen vector by ordinary recombinant DNA techniques to create a vectorwhich will produce expression of the protein of interest. For example, aDNA sequence encoding a hexose transporter protein may be used to createa hexose transporter (HX) vector. A population of the vector, e.g. theHX vector, may be obtained by expansion of the vector in bacterialculture, for vectors capable of replication in bacteria, or by PCR orother amplification techniques. The expanded population of vectors, e.g.HX vectors, is then used to transform a population of the chosen hostcells.

Transformation may be carried out by any suitable technique, includingelectroporation, DNA-coated microparticle bombardment, silica carbidewhisker vortexing, and glass bead vortexing. Preferably, glass beadvortexing or microparticle bombardment is used. More preferably,particle bombardment is used. Such techniques are well-known in the artand are described in greater detail herein and in WO 97/39106. See,also, U.S. Pat. No. 6,027,900 to Allnutt et al.

When microparticle bombardment using the Bio-Rad Biolisitc PDS-1000/HE™particle delivery system is employed (according to the manufacturer'sinstructions), preferably 1100, 1350, or 1500 psi rupture discs areused, although any psi rupture disc may be used. Most preferably, 1500psi rupture discs are used. Similarly, any microparticles commonly usedin the art may be used for bombardment. Preferably, tungsten particlesM5 (0.4 μm median diameter) or M17 (1.1 μm median diameter) or goldparticles of 1 micron median diameter are used. Most preferably,tungsten particles M17 are used. The cells to be bombarded may be placedon any level within the chamber. Preferably, the cells are placed onlevel two or three. Most preferably, the cells are placed on level two.Preferably, the DNA coated onto the microparticles is supercoiled.

In a preferred mode of the invention, successful transformants areselected in a first selection step, based on a characteristic of thevector such as resistance to an antibiotic, and only the successfultransformants are tested in a second step for the ability to growautotrophically using the substrate of interest (i.e., externalsource(s) of fixed carbon on which the transforming gene(s) shouldenable the transformed cells to grow). In another preferred mode, afterthe transformation step, cells are grown in reduced light in thepresence of low concentrations of the reduced carbon source whoseutilization is enabled by the transformation, e.g., sugar, and thetransformed host cells are subcultured into media with successivelyincreasing substrate concentrations to allow the transformed host toadapt to heterotrophic growth. Where the substrate of interest isglucose, the selection step may be testing the cells for ability to takeup ¹⁴C-glucose or for sensitivity to the toxic glucose analog,2-deoxyglucose. Preferably, light is withdrawn and the transformed cellsare tested for their ability to grow in the dark on the substrate ofinterest.

For example, DNA encoding a glucose transporter protein may be insertedinto a vector containing an antibiotic resistance gene, such asresistance to phleomycin. The resultant HX vector is used to transformalgal cells, and after the transformation step, the cells are plated outon media containing the antibiotic phleomycin and grown in the light.Colonies which appear represent cells that have acquired antibioticresistance as a result of successful transformation with the HX vector.These transformed cells are then plated out on media containing lowsugar concentration (e.g., 0.1% glucose). Colonies which grow in lowlight or in the dark on the low-sugar medium are replated onto mediacontaining a higher glucose concentration (e.g., 1% glucose). The cellswhich are able to grow in the dark on these high sugar plates areheterotrophic recombinant algae.

The trophic conversion method of this invention produces recombinantalgae capable of heterotrophic growth. These recombinant algae may begrown in fermentors to high cell numbers without the problem ofstarvation due to self-shading which occurs in phototrophic algae. Thus,algal cell products may be obtained in higher yield from fermentorculture of the recombinant algae of this invention which compared withthe yield from culture of the parent phototrophic algae. Further geneticmodification of the recombinant algae may be pursued by traditionalmutation methods or recombinant methods or both. Culturing the modifiedcells in the dark or in substantial darkness on substrate-containing(e.g., sugar) medium will maintain the heterotrophic nature of thecells, because the absence of light or substantial absence of lightprovides selective pressure suppressing cells losing the DNA encodingthe hexose transporter or other converting gene(s).

This invention also contemplates methods of using the recombinantheterotrophic algae for culturing in fermentors to produce desired algalproducts. Thus, this invention provides a method for obtaining algalbiomass or algal cell products comprising culturing in a fermentorrecombinant algal cells expressing, e.g., a heterologous hexosetransporter protein and harvesting the biomass and/or the desired algalcell product(s). Media and culture conditions for both plating out therecombinant algae and for fermentor culture may be readily determined bythe ordinary artisan through routine optimization. See, also, U.S. Pat.No. 5,244,921 to Kyle et al.; U.S. Pat. No. 5,374,657 to Kyle; U.S. Pat.No. 5,550,156 to Kyle; U.S. Pat. No. 5,567,732 to Kyle; U.S. Pat. No.5,492,938 to Kyle et al.; U.S. Pat. No. 5,407,957 to Kyle, et al.; U.S.Pat. No. 5,397,591 to Kyle et al.; U.S. Pat. No. 5,130,242 to Barclay,U.S. Pat. No. 5,658,767 to Kyle; and U.S. Pat. No. 5,711,933 to Kyle.

Products which may be produced by algae include, but are not limited to,pigments (e.g., β-carotene, phycobiliproteins, carotenolds,xanthophylls), oils with nutritional value (e.g., docosahexaenoic acid),and isotopically-labeled biochemicals (e.g., ¹³C- or ¹⁴C-glucose).

This invention also contemplates the use of algal biomass, either beforeor after extraction or partial extraction of desired products, as ananimal feed. A non-limiting example is the use of the biomass as anaquaculture feed for, e.g., larvae, ratifers, artemia, shrimp, fish,mollusks, vertebrates, or invertebrates. Other non-limiting examples arethe use of the biomass to feed, e.g., fowl, cattle, or pigs. The biomassmay also be used as a food or nutritional composition for humans.

The present inventors have also discovered that the methods of thepresent invention may be used as or to create a selection system fordetecting and/or selecting for transformed algae. These methods areparticularly useful in transformation of an organism which is able(either in its wild type state or through the heterotrophic conversionof the present invention) to grow on a given carbon source in the dark.Mutagenesis is used to disrupt a gene or genes necessary to and/orhelpful in uptake and/or catabolism of that carbon source. Means formutagenizing cells are well known in the art. Non-limiting,non-exclusive examples of methods for mutagenizing cells includechemical mutagenesis, radiation-induced mutagenesis, gene replacement,and site-directed mutagenesis.

Where gene replacement is used to inactivate the gene sought to beinactivated, a gene construct is produced containing the gene sought tobe inactivated with an antibiotic selectable marker inserted into themiddle of the coding region, resulting in a non-functional coding regionof the gene sought to be inactivated. The gene construct is theninserted into the cells by methods described herein. Where thenon-functional copy of the gene sought to be inactivated integrates andreplaces the endogenous; functional gene, the cells is rendered, e.g.,unable or less able to grow on the carbon source the catabolism of whichthe disrupted gene facilitated.

Cells which are unable or less able to grow on the carbon source may bedetected by various methods, such as assaying for the cells' ability totake up that carbon source or to grow on that carbon source in the dark.Where the gene which has been inactivated is a gene involved in glucoseuptake, the cells can easily be tested for inactivation of the gene ofinterest by growing them on the toxic glucose analog deoxyglucose. Cellspossessing a functional glucose transporter will import this compound.The deoxyglucose will enter the cells and not be metabolized. As aresult it will accumulate within the cells to toxic levels, killing thecells. Cells that can grow in the presence of deoxyglucose will likelynot be able to take up this compound, and, thus, they have beentransformed as desired. That the cells can not take up deoxyglucose orglucose can be confirmed by glucose uptake assays and the inability ofthe cells to grow in the dark on glucose as the sole carbon source.

Where a gene has been inactivated by gene replacement and an antibioticselectable marker has been inserted, cells with disrupted genes caneasily be selected for by growing the cells on the antibiotic, therebyselecting the resistant cells. Confirmation that the cells cannot takeup and/or catabolize a given carbon source can be tested by uptakeassays and/or assays for determining the cells' ability to grow on thatcarbon source in the dark.

A strain which has been rendered unable or less able to grow on a givencarbon source may then be transformed with other genes of interest inconjunction with transformation with a gene which will restore thecells' ability to grow in the dark on a particular carbon source. Inthis way, the ability to grow on a particular carbon source can be usedas a selectable marker for transformation, in a manner similar to thatin which antibiotic resistance is used as such a marker. Cells whichhave been successfully transformed may be selected by growing the cellsin the dark on the particular carbon source.

In a similar manner, the introduction of a gene or genes encodingprotein(s) that enable or enhance the growth of cells on a particularcarbon source on which they we are previously unable or less able togrow (as is described herein) may also be used as a selectable marker.In such a system, cells would be transformed with a gene of interest inconjunction with their transformation with a gene or genes encodingprotein(s) that enable or enhance the growth of cells on a particularcarbon source on which they were previously unable or less able to grow.

In this way, the ability to grow on a particular carbon source can beused as a selectable marker for transformation, in a manner similar tothat it which antibiotic resistance is used as such a marker. Cellswhich have been successfully transformed may be selected by growing thecells in the dark—on the particular carbon source.

Introduction or reintroduction of a gene encoding a protein enabling orenhancing uptake or growth on a particular carbon source would beaccomplished by methods described herein. The vector design would besimilar to that described herein, except that a gene encoding a proteinenabling or enhancing uptake or growth on a particular carbon sourcewould replace the antibiotic selectable marker. Guidance in carrying outtransformations and selection are provided herein.

As the cells of the present invention are able to grow heterotrophicallyand may be used as a transformation selection system, this inventioncontemplates the use of these cells, to produce recombinant protein. Insuch an application, autotrophic cells or cells the ability’ of which togrow in the dark on an external carbon source, has been disruptedthrough mutagenesis are transformed with a gene encoding the recombinantprotein sought to be made and a gene or genes encoding protein(s) thatenable or enhance heterotrophic growth. If desired, an antibioticselection system, as described herein, may be used to assist in theinitial screening of the cells for transformation. The cells which haveundergone heterotrophic transformation are then selected using themethods described herein. The selected cells are tested for theirability to produce the recombinant protein of interest. These cellswhich produce the protein can then be grown in fermentors underheterotrophic conditions, and the protein isolated therefrom usingtechniques which are well-known to those skilled in the art.

The trophic conversion of microalgae such as diatoms is a critical firststep in the engineering of algae for successful large-scale cultivationusing microbial fermentation technology. In addition to providing ameans for rigorously maintaining specific culture conditions with theobjective of maximizing productivity, the use of fermentation technologywill eliminate contamination of the cultures by microbes, which is animportant criterion for maintaining food industry standards as dictatedby the U.S. Food and Drug Administration. Furthermore, sugars such asglucose, as well as other growth-limiting nutrients, can be continuouslyprovided to the cultures such that growth rates remain saturated.Efficient fermentation has permitted the cultivation of the microalgaCrypthecodinium for production of the polyunsaturated fatty acid DHA foruse in human nutrition (Kyle, D J., (1996) Lipid Technology, 2:106).Optimizing conditions for fermentative growth of naturally heterotrophicalgae has resulted in dry biomass accumulation to 100 gm/liter (Gladue,et al., 1999; Running, et al., 1994) which is 10-50 fold higher than theyields obtained using light-dependent culture systems. Increased biomassaccumulation in fermentor systems results in production costs that areat least an order of magnitude less than those incurred using pondculture production methods (Radmer, et al., 1994). This reduced costincreases the feasibility for developing a large range of algalproducts, including polyunsaturated fatty acids (e.g. EPA), carotenoidsand xanthophyHs (e.g. (-carotene, lutein, filcoxanthin, astaxanthin),feeds for aquaculture and a variety of pharmaceuticals andnutraceuticals for market production. But the results also haveimportant implications with respect to fundamental biological aspects ofmarine ecosystems. Approximately 50% of the carbon fixation occurs inoceans, and the oceans serve as a major sink for fixed carbon (Raven, etal. (1999) Plant Cell and Environm., 22:741). The diatoms contributesubstantially to the reduction of inorganic carbon in marine habitats,and their contribution may increase substantially as the ecology ofoceanic environments are altered (M. R. Landry, et al., (2000) MarineEcology Progress Series, 201:57); B. Boyle, (1998) Nature 393:733;Takeda, (1998) Nature 393:777). The exploitation of diatoms that can begenetically manipulated and that can grow heterotrophically willfacilitate the use of mutants to augment our utilization of bothphotosynthesis and other metabolic pathways in algae that are essentialfor maintaining marine ecosystems.

EXAMPLES

In order to facilitate a more complete understanding of the invention, anumber of Examples are provided below. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only.

Example 1 Antibiotic Sensitivity in Diatoms

The diatoms Phaeodactylum tricornutum, Cylindrotheca fusiformis,Cyclotella cryptica and Nitzschia alba were extensively tested forsensitivity to a series of antibiotics for which resistance genes areavailable, and the lowest concentrations which completely abrogategrowth were determined for each antibiotic (Table C). Most of theantibiotics had no significant effect on growth or were effective onlyat very high concentrations compared to their effectiveness inabrogating growth of other eukaryotic organisms. These includeantibiotics such as G418, Hygromycin, Kanamycin and Spectinomycin, whichare routinely utilized for selection of other eukaryotic organisms.

For the diatoms tested, only the antibiotics zeocin and phleomycinresulted in cell death in 100% I.O. media (see Example 4 for descriptionof media and growth conditions).

It was found that by lowering the concentration of the media to 50% I.O.or less the sensitivity of the diatoms to antibiotics greatly increased.The effective concentration range of the antibiotic in most cases waslowered by a factor of 10. Representative diatoms were further testedagainst three of the more effective antibiotics at lower concentrationsof media.

Cells of each diatom species were plated on solid media at salinityequivalents of 25-100% I.O. media, with a gradient of antibioticconcentrations. After 2 weeks of illumination the growth of the cellswas assessed and the concentration of antibiotic required for cell deathwas determined. (Table D).

TABLE C Effect of antibiotics on the growth of selected diatoms. Valuesrepresent the lowest concentration of the antibiotic per ml required forcell death or the highest concentration tested. P. Antibioticstricornutum C. fusiformis C. cryptica N. alba Neomycin >1 mg >1 mg >1mg >1 mg Kanamycin >1 mg >1 mg >1 mg >1 mg Gentamicin >1 mg >1 mg >1mg >I mg Streptomycin >1 mg >1 mg >1 mg >I mg Spectinomycin >I mg >1mg >1 mg >1 mg G418 >1 mg >1 mg 250 mg >500 mg Hygromycin >1 mg 500mg >500 mg >500 mg Nourseothricin 250 mg >250 mg >250 mg >250 mgPuromycin 200 mg 100 mg 100 mg 100 mg Chloramphenicol 200 mg 100 mg 50mg 50 mg Erythromycin 100 mg 100 mg 50 mg 50 mg Bleomycin >50 mg >50 mgN.D. N.D. Phleomycin 5 mg 25 mg 25 mg >50 mg Zeocin 50 mg >250 mg 250mg >250 mg

TABLE D Effect of antibiotics on the growth of selected diatoms in mediawith reduced salinity. The relative percentage of salinity verses normalseawater of the medium is given in parentheses. Antibiotic levels aregiven as the lowest concentration of antibiotic per ml required for celldeath or the lowest concentration tested. P. tricornutum C. fusiformisC. cryptica Zeocin 25 mg (50%) 250 mg (50%) <5 mg (10%) 100 mg (25%)Nourseothricin 25 mg (50%)  50 mg (50%) <5 mg (10%) 10 mg (25%) <10 mg(25%) Phleomycin <5 mg (50%)  25 mg (50%) <5 mg (10%) <5 mg (25%)  <5 mg(25%)

Example 2 P. tricornutum is Incapable of Heterotrophic Growth

To confirm that P. tricornutum was incapable of heterotrophic growth onglucose, cells were repeatedly plated on glucose containing solid mediaand placed in the dark. Cells typically divided up to 1-2 times over 24hrs, then stopped growth. No additional divisions were evident after 1-2months. Cultures were also checked for glucose transporter activity. Nodetectable uptake was evident even after 4 hours of incubation. Cellswere also plated on 16 different hexose or related sugars (arabiose,cellobiose, fructose, fucose, galactose, gluconate, lactose, maltose,maltobiose, mannose, meliblose, nibose, sorbitol, suclose, trehalose,xylose), with no detectable growth.

Example 3 Generic Mutation does not Enable Heterotrophic Growth of P.tricornutum

To confirm that P. tricornutum is not capable of spontaneous mutationsthat result in the ability to grow in the dark on glucose, 10¹⁰ controlcells were plated onto glucose-containing solid media. No spontaneouscolonies formed on glucose. To confirm that the transformation procedureand/or insertion of foreign DNA could not result in trophic conversion,approximately 100 cell lines were transformed with pPha-T1 containing avariety of heterologous genes (uidA gfp, nat, and nptlI), but not glutlor hupl, and these transformed cell lines were tested for growth onglucose in the dark. None grew on glucose.

Example 4 Transformation of P. tricornutum for Heterotrophic Growth

Phaeodactylum tricornutum is one microalga that can be geneticallymodified by transformation (Zaslayskaia, et al. (2000), Apt, et al.(1996)), but that is unable to grow heterotrophically (Cooksey, 1974;Droop, 1974; Hellebust, et al., 1977). A trophic conversion of this algawas attempted by transforming it with genes encoding glucosetransporters. The glucose transporter genes used for transformationincluded Glut1, from human erythrocytes (Mueckler, et al., 1997), Hup1from Chlorella kessleri (Sauer, et al., 1989), and Hxt1, Hxt2, Hxt4 fromSaccharomyces cervisiae (Kruckeberg, 1996). The coding regions of thesegenes were inserted into the P. tricornutum transformation vectorpPha-T1, which uses the promoter of a gene encoding the fucoxanthinchlorophyll binding proteins (Fcp) to drive expression of foreign genesin diatoms (Barclay, et al., 1994; Zaslayskaia, et al., 2000). Aconstruct was also generated in which the GFP gene was fused to the 3′end of the Glut gene. Plasmids were introduced into P. tricornutum usingbiolistic procedures and transformants were selected for Zeocinresistance in the light (Zaslayskaia, et al., 2000). The transformantswere then transferred to solid or liquid medium containing 0.1 or 1.0%glucose, placed in complete darkness and monitored for growth.

Culture Conditions: Phaeodactylum tricornutum Bohlin (University ofTexas Culture Collection, strain 646) was grown at 20° C. withcontinuous illumination at 75 μmol photons M⁻²s⁻¹ in Provasoli'senriched seawater medium made with Instant Ocean™ (I.O.) artificialseawater, instead of natural sea water, at 0.5× concentration. See,Stair, R. C. & Zeikus, J. A. (1993) “UTEX—The culture collection ofalgae at the University of Texas at Austin,” J. Phycol. 29 (Suppl):93.Solid medium contained 1.2% agar and liquid cultures were bubbled withair containing 1% CO₂ in Roux bottles.

Constructs: The sequence of the Phaeodactylum transformation vectorpPha-TI (see FIG. 1) has the Genbank accession No. AF219942. The fcpApromoter has been placed in front of the multiple cloning site (MCS).The fcpB promoter was placed in front of the sh ble gene. The constructalso contains the ampicillin resistance gene (Amp) and the E. coliorigin of replication.

pPha-T1 (FIG. 1) was constructed in several stages. The first stepinvolved subcloning the fcpA terminator region from pfcpB/ble (Apt etal. 1996) into the HindIII-XhoI sites of pSP73 (Promega). The zeocinresistance cassette from pfcpB/ble (Apt et al. 1996), which contains thefcpB promoter driving the sh ble gene, was subcloned as a XhoI fragmentinto the pSP73 XhoI site. The fcpA promoter region from pfcpB/ble (Aptet al. 1996) was subcloned as a PstI-EcoRI fragment and inserted intoBluescript SK−. This same fragment was then removed as a BamHI-EcoRVfragment and ligated into the BgIII-EcoRV sites of pSP73 to form thefinal basic construct. The multi-cloning site from pSP73 between theEcoRV and HindIII sites was preserved intact, excluding the ClaI site,which was removed by site-directed mutagenesis (Deng and Nickoloff 1992)to eliminate a cryptic ATG start codon. The pPha-T1 vector contains tencommonly used, unique restriction sites into which genes of interest canbe inserted.

A series of glucose transporters were tested for expression in P.tricornutum. These include the hxt1, hxt2, htx4 genes from Saccromycescerviesae, the Chlorella kessleri hup1 gene, and glutl found inerythrocytes. Kruckeberg A L (1996) “The hexose transporter family ofSacchoromyces cerevisisae,” Arch. Microbial. 166:283-92; Sauer N, TannerW (1989) “The hexose carrier from Chlorella cDNA cloning of a eucryoticH+-cotranporter,” FEBS Lett 259:43-46; Mueckler M, Hresko R C, Sato M(1997) “Structure, function and biosynthesis of GLUTI,” BiochemicalSociety Transactions 25:951-4. The coding regions of these genes wereinserted into the transformation vector pPha-T1.

Plasmids used for construction of transformation vectors used tointroduce glucose transporters into cells of Phaeodactylum wereconstructed by adding appropriate restriction sites by PCR and insertingthe coding region for the transporter of interest into pPha-T1. PlasmidpGlut-Pkat used primers GLUTPHAT5′ (GACTGGATCCATGGAGCCCAGCAGCAAG) andGLUTPATT3′ (GACTAAGCTT-TCACACTTGGGAATCAGC). Plasmid pHup-Phat usedprimers HUPPHAT5′ (GATGAATTCA-TGGCCGGCGGTGGTGTAG) and HUPPHAT3′(GACTAAGCTTTTACTTCATCGCCTTTGAC). Plasmid pHxt2-Phat used primersHXT2PHAT5′ (GGGAATTCATTCAAGATGTC-TGAGTTCGCTAGAAG) and HXT2PHAT3′(CCCCGCATGCTTATTCCTCGGAAACTCTT Plasmid pHxt4-Phat used primersHXT4PHAT5′ (GGGAATCATTCAGGATGTCTGAAGAAGCT) and HXT4PHAT3′(CCTCTAGATTACTTTTTTCCGAACAT C).

Microparticle Bombardment: Cells were bombarded with the transformationvector containing the gene of interest using the Bio-Rad BiolisticPDS-1000/He Particle Delivery System fitted with 1,500 psi rupturediscs. Tungsten particles M17 (1.1 μm median diameter) were coated with0.8 mg plasmid DNA in the presence of CaCl2 and spermidine, as describedby the manufacturer. Approximately 5×10⁷ cells were spread in the centerone third of a plate of solid 0.5×I.O. medium 1 h prior to bombardment.The plate was positioned at the second level within the Biolisticchamber for bombardment. Bombarded cells were illuminated for 24 h(cells divided once during this period) prior to suspension in 0.5 ml ofsterile 0.5×I.O. medium; 100 μL of this suspension (˜1×10⁷ cells) wasplated onto solid medium containing 100 μg/ml Zeocin. The plates wereplaced under constant illumination (75 μmol photons m⁻²s⁻¹) for 2-3weeks and resistant colonies were re-streaked on fresh solid mediumcontaining at least 100 μg/ml Zeocin.

Colonies of primary transformants were restreaked on 250 μg/ml Zeocin.After two weeks cells were restreaked on media containing 0.1% and 1.0%glucose, and were kept in darkness by wrapping with several layers offoil. After 4 weeks transformants that had detectable growth wererestreaked and maintained on 1.0% glucose. Liquid cultures were grown in10 media with 1.0% glucose at 20° C. on an orbital shaker.

Each zeocin resistant cell line was also checked for glucose uptake. Allof these transformants resulted from independent particle bombardments.

Glucose Uptake, and Growth on Glucose: 250-500 ml of transformedPhaeodacrylum cells in logarithmic phase growth were harvested at 2000rpm for 10 min, washed 2 times with 50% I.O., resuspended in I.O. andcounted. Cells were aliquoted into 50 ml tubes (7 ml/tube). Unlabelledglucose was added from a stock solution of 0.1M in 50% I.O. to theappropriate concentration. D-¹⁴C-glucose (ICN) was added to aconcentration of 0.05 μCi per mL. Samples (1 ml) were taken at minintervals, cells were filtered on nitrocellulose and washed 3 times with50% I.O. containing 5% unlabeled glucose. The filters were transferredto scintillation vials with 10 ml of Scintisafe (Fisher), incubated for1 hr and counted. Heat-killed or aldhyde fixed cells were used ascontrols.

For glut1 containing transformants, 22 out of 32 zeocin resistant celllines were capable of detectable glucose uptake. Uptake rates rangedfrom a high of 8.8 to 2.0 nmole·(10⁸ cells·min)⁻¹ (Table E). Cells lineswith uptake rates of 1.6 nmole glucose (10⁸ cell·min)⁻¹ or greater (11of 28) were able to grow on glucose in the dark. For hup1 containingtransformants 14 out of 25 antibiotic resistant cell lines were capableof glucose uptake. The uptake rates ranged from 1.72 to 0.06 nmole·(10⁸cells·min.)⁻¹ (Table F). Cell lines with uptake rates of 0.29 mmole (10⁸cells·min.)⁻¹ or greater (11 of 25) were able to grow in the dark. Noneof the P. tricornutum transformants transformed with vectors containingthe yeast transporters were capable of detectable glucose uptake. Thepresence of the hxt 1, 2, or 4 genes or the gene products within cellswas not confirmed. The inability of the transformants to expressfunctional Hxt protein may reflect striking differences in codon usagebetween yeast and P. tricornutum (Zaslayskaia, et al., 2000).

Eleven of the glut1 and 11 of the hupl containing transformants haddetectable growth on agar plates or in liquid media containing 0.1%glucose after 2-4 weeks in complete darkness. GlutI containingtransformants with uptake rates greater than 2.0 mmole·(10⁸cells·min.)⁻¹ were all capable of growth in the dark on glucose. AllHup1 containing transformants with glucose uptake rates greater than0.29 nmole·(10⁸ cells·min.)⁻¹ cells were capable of heterotrophicgrowth.

To confirm that the transformation procedure and/or insertion of foreignDNA did not result in trophic conversion, approximately 100 cell linestransformed with pPha-T1 containing a variety of heterologous genes(uidA gfp, nat, and nptII), but no glucose transporters, were plated onglucose (0.1%) containing solid media. None of these cell lines haddetectable grow after 4 weeks in the dark.

Heterotrophic growth rates were determined for transformants Glut-13,Glt-17, and Hup-2. All three cell lines had division rates ofapproximately 0.7 divisions per day.

TABLE E Glucose uptake rates of glutl containing transformants of P.tricornutum. Strain Uptake Rate Growth Glut-28 8.8 + Glut-13 7.6 +Glut-26 5.9 + Glut-25 4.9 + Glut-17 4.6 + Glut-34 3.9 + Glut-11 3.5 +Glut-21 3.2 + Glut-15 2.6 + Glut-1 2.4 + Glut-32 1.9 − Glut-24 1.8 −Glut-3 1.6 + Glut-9 1.6 + Glut-12 1.4 − Glut-3 0.94 − Glut-7 0.93 −Glut-19 0.90 − Glut-22 0.82 − Glut-2 0.48 − Glut-20 0.25 − Glut-10 0.20− Glut-4 0.0 − Glut-5 0.0 − Glut-6 0.0 − Glut-14 0.0 − Glut-18 0.0 −Glut-23 0.0 − Glut-29 0.0 − Glut-30 0.0 − Glut-31 0.0 − Glut-33 0.0 −Uptake rates are expressed as nmole * (10⁸ cells * min.)⁻¹. Cells linescapable of heteratrophic growth are designated with a “+” cells that hadno growth are designated as “−”.

TABLE F Glucose uptake rates of hup1 containing transformants of P.tricornutum. Strain Uptake Rate Growth Hup-2 1.72 + Hup-12 1.40 + Hup-151.40 + Hup-23 1.40 + Hup-24 0.94 + Hup-4 0.84 + Hup-8 0.70 + Hup-100.68 + Hup-18 0.53 + Hup-13 0.43 + Hup-9 0.29 + Hup-6 0.26 − Hup-20 0.24− Hup-25 0.06 − Hup-1 0 − Hup-3 0 − Hup-5 0 − Hup-7 0 − Hup-11 0 −Hup-14 0 − Hup-16 0 − Hup-17 0 − Hup-19 0 − Hup-21 0 − Hup-22 0 − Uptakerates are expressed as nmole * (10⁸ cells * min.)⁻¹. Cells lines capableof heterotrophic growth are designated with a cells that had no growthare designated as “−”.

Example 5 Incorporation of Desired Genetic Inserts

Cells lines Glut-17 and Hup-2 were used to examine the integration andexpression of the respective glucose transporters. Based on Southernblots (see, FIG. 2), Glut-17 appears to have one copy of glut1 and Hup-2probably has multiple copies of hup1. RNA blots (see, FIG. 3) indicatethat each cell line produces a transcript of the expected size.

Preparation of Nucleic Acids: for the Preparation of Total DNA, theCells were pelleted by centrifugation (5,000×g for 10 min), lysed in 50mM Tris-HCl, pH 8.0, 10 mM EDTA, 1.0% SDS, 10 mM DTT, 10 μg/ml proteaseK, 20 μg/ml RNAse A and incubated at 37° C. for 15 min. The lysate wasextracted with one volume of phenol:chloroform (1:1) and again with onevolume of chloroform. The aqueous phase was collected and made 1.2 g/mlCsCl and 0.2 mg/ml ethidium bromide prior to centrifugation in a BeckmanV663.2 rotor for 6 h at 55,000 rpm. The DNA band was collected,extracted with butanol, precipitated with 2 volumes of ethanol andresuspended in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) to a finalconcentration of 1 mg/ml. RNA was extracted using the method ofChomczynski, (1994, “Single-step method of total RNA isolation by acidguanidine-phenol extraction.” In: Celis J. E. (ed) Cell Biology: alaboratory handbook, Vol I. Academic Press, San Diego, pp 680-683).

Gel Electrophoresis and Hybridization Conditions: DNA was resolved onagarose gels in TAE buffer (Maniatis et al. 1982). Nucleic acids weretransferred to Nytran filters (Schleicher and Schuell) and cross-linkedwith UV light using a Stratalinker (Stratagene). Hybridizations wereperformed in a Bachofer rotary oven at 65° C. overnight in 5×SSFE, 1%SDS, 5×Denhardt's and 100 μg/ml DNA, according to standard protocols(Sambrook et al. 1989). Following hybridizations, the filters werewashed three times for 30 min at the hybridization temperature in 50 mMphosphate buffer containing 0.1% SDS. The filters were then dried andexposed to X-ray film for 1 to 4 d (Kodak XAR5). Total RNA was resolvedon an agarose gel containing formaldehyde according to the method ofRosen, et al. (1990, “Optimizing the Northern blot procedure,”Biotechniques 8:398-403).

Example 6 Localization of the Protein Expressed from the Inserted Gene

A more detailed characterization was performed for a number of the Gluttransformants, including Glut-17 and Glut GFP-40. The latter strain wastransformed with pPha-T 1 harboring the glut1 gene fused to GFP.Transformed cells were broken using a MinibeadBeater by two breakagecycles at full speed (30 sec for each cycle with 3-5 mm cooling on icebetween cycles), and the membranes were pelleted by centrifugation at100,000×g for 30 and then solubilized in 2% SDS. The solubilizedproteins were resolved on a 7.5% polyacrylamide gel, transferred tonitrocellulose membranes (Towbin, et al. (1979) Proc. Natl. Acad. Sci.USA, 76:4350) and the Glut1 or GFP proteins were detectedimmunologically (Liscum, et al., (1995) Plant Cell, 7:473). Monospecific antibodies against the Glut1 polypeptide and GFP were used todemonstrate accumulation of Glut1 or the Glut1GFP fusion protein intransformed cell lines.

Membranes of the Glut-17 transformant contained two prominentpolypeptides that reacted with Glut1-specific antibodies (FIG. 4). Thesepolypeptides had molecular masses of 44 and 39 kDa, which is less thanthat of the native protein (approximately 55 kDa) synthesized in humanerythrocytes (FIG. 4, compare lanes B and G1-17), but which is close tothe size of unglycosylated Glut1 (38 kDa) (Asano, et al. (1991) J. Biol.Chem., 266:24632). This implies that the Glut1 that accumulates in P.tricornutum is glycoslated differently than in human erythrocytes. TheGlut1GFP fusion protein present in the Glut1 GFP-40 transformant had amolecular mass of approximately 75 kDa, which is also slightly smallerthan the expected size of Glut1GFP (82 kDa).

To determine the subcellular location of the Glut 1 protein intransformed lines, the Glut1GFP-40 strain was examined for GFPfluorescence by confocal microscopy. Cells were gently smeared ontocoverslips (#1½) and mounted in a thin layer of artificial seawater.Confocal microscopy was performed using a Nikon 60×N.a.=1.2 waterimmersion objective on a Nikon TMD 200 inverted microscope outfittedwith a BioRad MRC 1024 confocal head mounted in a Koehler configuration.EGFP was excited at 488 nm and visualized with a 522/25 nm bandpassfilter. Plastid autofluorescence was excited at 456 nm and visualizedwith a 585 mm long pass filter. Images were adjusted in Adobe Photoshopsuch that control and experimental images were treated identically. Onlylinear adjustments of contrast and brightness were performed on theoriginal images.

While nontransformed cells showed strong chlorophyll fluorescence in thered fluorescence channel, only a small amount of fluorescence wasobserved in the green channel (FIG. 6, top panel). Cells transformed toexpress GFP from the vector pPha-T1 exhibited intense GFP fluorescencein a pattern consistent with localization in the cytosol and the lumenof the cell nucleus (FIG. 6, lower left). A similar distribution ofsoluble GNP in plant cells has been observed (Chiu, et al. (1996) Curr.Biol., 6:325; Haseloff, et al. 1997) Proc. Natl, Acad. Sci. USA,94:2122). In contrast, when the Glut1GFP chimeric construct isintroduced into diatom cells the majority of the fluorescence isassociated with the extreme periphery of the cells (FIG. 6, lowerright). These results demonstrate that the Glut 1 protein targets GFP tothe cell cortex, a pattern consistent with localization of the chimericprotein to the cytoplasmic membrane and the function of Gluti as acytoplasmic membrane-associated transporter.

Example 7 Kinetics of Glucose Uptake by Transformed Cells

Detailed glucose uptake kinetics were done on various glut containingtransformants, which grew well on glucose containing media. 250-500 mlof transformed P. tricornutum cells in logarithmic phase growth wereharvested at 4,500 rpm (SA600 rotor) for 10 mm, washed 2 times,resuspended in and counted all with 50% 1.0. medium. Assays wereinitiated by adding unlabelled glucose (to the appropriateconcentration) from a stock solution of 0.1M in 50% I.O. andD-¹⁴C-glucose (ICN) to 0.05 μCi per mL; the cells were maintained in thelight during the assay. Samples (800 μl) were withdrawn from the assaymixture at specific time intervals (0, 2, 5, 10 and 15 mm) following theaddition of the labeled glucose. The cells were filtered onto Supor(polyethersulfone) membranes (Gelman Scientific) and washed with 50%I.O. medium containing 1% unlabeled glucose. The membranes weretransferred to scintillation vials with 5 ml of Scintisafe (Fisher),incubated for 1 h, at 20° C. and then counted.

The Glut-13, Glut-17 and Glut GFP-40 transformants showed high rates ofglucose uptake (FIG. 5). Transformant Glut-13 had a Km of 1.2 mM and aVmax of 1.4-3 nmoles glucose (10⁸ cells·min)⁻¹. Glut-17 had a Km of 0.9mM and a Vmax of 1-5 nmoles glucose (10⁸ cells·min)⁻¹. The Glut-17transformant had a Km for glucose of 1.2 mM and a Vmax of 7.6 nmolesglucose (10⁸ cells·min)⁻¹ while the Glut]GFP-40 transformant had a Km ofapproximately 1.0 mM and a Vmax of 13 nmoles glucose (10⁸ cells·min)⁻¹.The Km values for glucose in the transformants are similar to those (1-2mM) measured for human erythrocytes (Chisholm, S. W., (2000) Nature,407:685; mueckler, et al., 1997). Differences in the Vmax probablyreflect different levels of expression of the Glut1 gene, which woulddepend on the site of integration into the diatom genome. To furtherconfirm that the glut1 transporter is functioning normally the specificinhibitor Cytochalasin B was incubated with the cell line Glut-13.Cytochalasin B is well known to be a specific inhibitor of type glucosetransporters (Lu, et al., (1997). J. Chromatog., 776:81). In thepresence of 5×10⁻⁴ of Cytochalasin B glucose uptake was reduced to anundetectable level. The inhibitor data further support that theheterologous Glut1 transporter is functioning in the correct manner.

The glucose uptake kinetics for transformant Hup-2 was also measured indetail with a Km of 40 μM and a Vmax of 3 nmoles glucose (10⁸cells·min)⁻¹. The Km values are slightly higher than the publishedvalues measures for intact Chlorella cells and the Hup1 produced inyeast or Xenopus, which were 10-20 μM (Sauer N, Caspari T, Klebl F,Tanner W (1990) “Functional expression of the Chlorella hexosetransporter in Schzosaccharomyces pombe, Proc. Natl Acad. Sci.87:7949-52; Aoshima H, Yamada M, Sauer N, Kornor E, Schobert C (1993)“Heterologous expression of the H+/hexose cotransporter from Chlorellain Xenopus oocytes and its characterization with respect to sugarspecificity, pH and membrane potential,” J. Plant Physiol. 141:293-297).

Example 8 Comparison of Light and Dark Growth for a Transformant

Growth of the Glut-17 transformant was measured in the light and dark inmedium supplemented with glucose. Glucose levels were typicallymaintained between 5-10 g/L. Growth rates were monitored in culturesmaintained in 50 mL of medium in 250 mL flasks with silicon foamclosures. Samples were taken on a daily basis to measure cell numbersand nutrient levels. Flasks were shaken on a rotary platform at 100 rpm.High density cultures were grown in a 2 L Applikont fermentor using anagitation rate of 100 rpm, dissolved oxygen was maintained at >20%saturation and the pH was at 7.5.

As shown in FIG. 7, both untransformed cells and the Glut-17transformant grown in the light reached approximately the same celldensities (2×10⁷ cells·ml⁻¹). The addition of glucose to the medium didnot change the growth characteristics of the untransformed strain. Incontrast, the transformed strain attained a cell density that wasapproximately 5 times higher than that of the untransformed cells (overa five-day growth period). Furthermore, while untransformed cells areunable to grow in the dark in the presence of glucose, the Glut-17transformant grows at the same rate and to the same cell density as whenit is grown in the presence of glucose in the light. Strikingly, as thecultures become more dense and light absorption is attenuated byself-shading, the rate of growth of the transformant in the presence ofglucose exceeds that of untransformed cells. If heterotrophic growth isconducted in a microbial fermentor with continuous addition of glucoseand other nutrients to the medium, the density attained by cultures ofthe transformant can exceed that of wild-type cells by 10 to 20 fold,reaching densities of 5×10⁸ cells·mL⁻¹.

Example 9

Mutated cells are produced by exposing cells to U.V. light (210-260 nm)at levels sufficient to result in >90% cell death.

Example 10

Mutated cells are produced by mixed cells in liquid withNitrosoguanidine (a chemical mutagen) for 5 min at sufficientconcentration to cause >90% cell death. The chemical mutagen is washedfrom the cells and the cells plated on solid media.

Example 11

The gene encoding the glucose transporter in a group of cells isinactivated by a gene replacement. A gene construct is producedcontaining the glucose transporter endogenous to the cells, with a thezeocin resistance cassette (described herein) inserted into the centerof the transporter coding regions, resulting in a nonfunctional codingregion of the glucose transporter. The gene construct is introduced intothe cells using microparticle bombardment. This gene integrates andreplaces the functional endogenous glucose transporter. Cells are grownon zeocin. Cells able to grow on zeocin are selected for further assaysto determine whether the endogenous glucose transporter has beeninactivated.

Example 12

Cells which have been mutagenized, as, for example, in Examples 9, 10,or 11, are grown in the presence of deoxyglucose. Cells which can groware unable to take up the toxic deoxyglucose, which indicates that theydo not possess functional glucose transporters. Cells without afunctional glucose transporter are selected for further experiments.

A gene encoding a glucose transporter is inserted into the selectedcells via the methods described herein. In conjunction with thistransformation, the cells are also transformed with a second gene (“agene of interest”).

Cells are tested for their ability to grow on glucose in the dark, asdescribed herein. Cells which can grow on glucose in the dark,indicating the expression of a functional glucose transporter, areselected for further-testing to determine whether they have also beentransformed by the gene of interest.

The practice of the present invention employs, unless otherwiseindicated, conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques are wellknown to the skilled worker and are explained fully in the literature.See, e.g., Sambrook, J., Maniatis, T., & Fritsch, E. F. (1939),Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y.; “DNA Cloning: A Practical Approach,”Volumes I and II (D. N. Glover. ed., 1985); “Oligonucleotide Synthesis”(M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S.J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames &S. J. Higgins, eds., 1984); “Animal Cell Culture”.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein, however, is notto be construed as limited to the particular forms disclosed, since theyare to be regarded as illustrative rather than restrictive. Variationsand changes may be made by those skilled in the art without departingfrom the spirit of the invention.

For purposes of clarity of understanding, the foregoing invention hasbeen described in some detail by way of illustrations and examples inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons of skill in molecularbiology, phycology, and/or related fields are intended to be within thescope of the invention, which is limited only by the appended claims.

All publications and patent applications mentioned in this specificationare incorporated herein by reference to the same extent as if they hadeach been individually incorporated by reference. All publications andpatent applications mentioned in this specification are indicative ofthe level of skill of those skilled in the art to which this inventionpertains.

1-22. (canceled)
 23. A method of producing an algal biomass comprisingculturing Bacillariophyta alga cells capable of growing in the absenceof light to produce a biomass, wherein Bacillariophyta alga cells in theculture comprise an exogenous transgene, wherein the transgene comprisesa nucleic acid construct encoding a glucose transporter selected fromthe group consisting of Glut1 (human erythrocyte glucose transporter 1)and Hup1 (Chlorella HUP1 Monosaccharide-H+ Symporter) under the controlof a functionally linked promoter, wherein upon expression of theglucose transporter in an amount sufficient to transport glucose intothe Bacillariophyta alga cells, the Bacillariophyta alga cells grow onglucose in the absence of light as compared to untransformed wild-typeBacillariophyta alga cells, and wherein the untransformed wild-typeBacillariophyta alga cells are obligate photoautotrophs.
 24. The methodof claim 23, wherein the Bacillariophyta alga cells are selected fromthe group consisting of Nitzschia, Navicula, Thalassiosira, andPhaeodactylum cells.
 25. The method of claim 24, wherein thePhaeodactylum cells are Phaeodactylum tricornutum cells.
 26. The methodof claim 23, wherein the promoter is a light harvesting promoter. 27.The method of claim 26, wherein the light harvesting promoter is afucoxanthin chlorophyll binding protein (fcp) promoter.
 28. The methodof claim 27, wherein the fop promoter is fcpA, fcpB fcpC, or fcpE. 29.The method of claim 23, wherein the method further comprises harvestingthe biomass.
 30. A biomass produced by the method of claim
 23. 31. Ananimal feed comprising the biomass of claim
 30. 32. A method ofproducing an oil, comprising extracting an oil from the biomass of claim30.
 33. A method of producing a pigment, comprising extracting a pigmentfrom the biomass of claim
 30. 34. The method of claim 33, wherein thepigment is selected from the group consisting of β-carotene, aphycobiliprotein, a carotenoid, and a xanthophyll.
 35. A method for theheterotrophic conversion of Bacillariophyta alga cells, comprising: (a)transforming Bacillariophyta alga cells with an exogenous transgene,wherein the transgene comprises a nucleic acid construct encoding aglucose transporter selected from the group consisting of Glut1 (humanerythrocyte glucose transporter 1) and Hup1 (Chlorella HUP1Monosaccharide-H+ Symporter) tinder the control of a functionally linkedpromoter, wherein upon expression of the glucose transporter in anamount sufficient to transport glucose into the Bacillariophyta algacells, the Bacillariophyta alga cells grow on glucose in the absence oflight as compared to untransformed wild-type Bacillariophyta alga cells,and wherein the untransformed wild-type Bacillariophyta alga cells areobligate photoautotrophs, and (b) selecting transformed alga cells bygrowing the cells on glucose in the absence of light.
 36. The method ofclaim 35, wherein the Bacillariophyta alga cells are selected from thegroup consisting of Nitzschia, Navicula, Thalassiosira, andPhaeodactylum cells.
 37. The method of claim 36, wherein thePhaeodactylum cells are Phaeodactylum tricornutum cells.
 38. The methodof claim 35, wherein the promoter is a light harvesting promoter. 39.The method of claim 38, wherein the light harvesting promoter is afucoxanthin chlorophyll binding protein (fcp) promoter.
 40. The methodof claim 39, wherein the fcp promoter is fcpA, fcpC, or fcpE.
 41. Themethod of claim 35, wherein the method further comprises transformingthe Bacillariophyta alga cells with a gene of interest.
 42. The methodof claim 41, wherein the gene of interest encodes a recombinant protein.43. A transformed alga cell produced by the method of claim
 41. 44. Amethod of producing a recombinant protein comprising culturing thetransformed alga cell of claim 43 under conditions allowing theexpression of the gene of interest, wherein the gene of interest encodesa recombinant protein, to produce the recombinant protein.